Molecular mapping the presence of druggable targets in preinvasive and precursor breast lesions: A comprehensive review of biomarkers related to therapeutic interventions

The analysis of clinical breast samples using biomarkers is integral to current breast cancer management. Currently, a limited number of targeted therapies are standard of care in breast cancer treatment. However, these targeted therapies are only suitable for a subset of patients and resistance may occur. Strategies to prevent the occurrence of invasive lesions are required to reduce the morbidity and mortality associated with the development of cancer. In theory, application of targeted therapies to pre-invasive lesions will prevent their progression to invasive lesions with full malignant potential. The diagnostic challenge for pathologists is to make interpretative decisions on early detected pre-invasive lesions. Overall, only a small proportion of these pre-invasive lesions will progress to invasive carcinoma and morphological assessment is an imprecise and subjective means to differentiate histologically identical lesions with varying malignant potential. Therefore differential biomarker analysis in pre-invasive lesions may prevent overtreatment with surgery and provide a predictive indicator of response to therapy. There follows a review of established and emerging potential druggable targets in pre-invasive lesions and correlation with lesion morphology.     

A 5q12.1–5q12.3 microdeletion in a case with a balanced exceptional complex chromosomal rearrangement

Complex chromosomal rearrangements are very rare chromosomal abnormalities. Individuals with a complex chromosomal rearrangement can be phenotypically normal or display a clinical abnormality. It is believed that these abnormalities are due to either microdeletions or microduplications at the translocation breakpoints or as a result of disruption of the genes located in the breakpoints. In this study we describe a 2-year-old child with mental retardation and developmental delay in whom a de novo apparently balanced exceptional complex chromosomal rearrangement was found through conventional cytogenetic analysis. Using both cytogenetic and FISH analysis, the patient's karyotype was found to be: 46,XY,der(5)t(5;7)(p15.1;7q34),t(5;8)(q13.1;8q24.1)dn. A large, clinically significant deletion which encompassed 887.69 kb was detected at the 5q12.1–5q12.3 (chr5:62.886.523–63.774.210) genomic region using array-CGH. This deleted region includes the HTR1A and RNF180 genes. This is the first report of an individual with an apparently balanced complex chromosomal rearrangement in conjunction with a microdeletion at 5q12.1–5q12.3 in which there are both mental-motor retardation and dysmorphia.     

Genetic analysis of ORF5 porcine reproductive and respiratory syndrome virus isolated in Vietnam

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important swine pathogens because it is highly infectious and causes economic losses due to decreased pig productivity. In this study, the 603 bp complete major envelope protein encoding gene (ORF5) of 32 field PRRSV isolates from Vietnam collected during 2008–2012 were sequenced and analyzed. Multiple nucleotide (nt) and deduced amino acid (aa) alignments of ORF5 were performed on the 32 isolates: the representative strains (European and North American genotypes), Chinese strains available in GenBank and vaccine strains licensed for use in Vietnam. The results showed 94.8–100.0% nt identity and 94.0–100% aa similarity among the 32 isolates. These isolates shared similarities with the prototype of the North American PRRSV strain (VR‐2332; nt 87.8–89.3%, aa 87.5–90.0%), and Lelystat virus, the prototype of the European PRRSV strain (LV; nt 61.1–61.9%, aa 55.1‐57.0%). There was greater similarity with QN07 (nt 96.5‐98.5%, aa 96.0‐99.0%) from the 2007 PRRS outbreak in QuangNam Province, CH‐1a (nt 93.2–95.1%, 91.5–93.5%) isolated in China in 1995 and JXA1 (nt 96.5–98.6%, aa 95.0–98.0%), the highly pathogenic strain from China isolated in 2006. The Vietnamese isolates were more similar to JXA1‐R (nt 96.5–98.6%, aa 95.0–98.0%), the strain used in Chinese vaccines, than to Ingelvac MLV/BSL‐PS (nt 87.2–89.0%, aa 86.0–89.0%). Phylogenetic analysis showed that the 32 isolates were of the North American genotype and classified into sub‐lineage 8.7. This sub‐lineage contains highly pathogenic Chinese PRRSV strains. This study documents genetic variation in circulating PRRSV strains and could assist more effective use of PRRS vaccines in Vietnam.     

Interactions of Meso-tetra-(4-N-oxyethylpyridyl) Porphyrin,Its 3-N Analog and Their Metallocomplexes with Duplex DNA

Abstract

Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin (TOEPyP(4)), its 3-N analog (TOEPyP(3)) and their Co, Cu, Ni, Zn metallocomplexes with duplex DNA have been investigated by uv/visible absorbance and circular dichrosim spectroscopies. Results reveal the interactions of these complexes with duplex DNA are of two types. (1) External binding of duplex DNA by metalloporphyrins containing Zn and Co, and (2) Binding of duplex DNA both externally and internally (by intercalation) by porphyrins not containing metals, and metalloporphyrins containing Cu and Ni. Results indicate that (4N-oxyethylpyridyl) porphyrins intercalate more preferably in the structure of duplex DNA and have weaker external binding than 3N-porphyrins.     

Hospitalization-induced exacerbation of the ill effects of chemotherapy on rest-activity rhythm and quality of life of breast cancer patients: a prospective and comparative cross-sectional follow-up study

ABSTRACT

Chemotherapy administration may result in the disruption of circadian rhythms and impairment of quality of life (QoL) of cancer patients. Nevertheless, we have little knowledge on the long-term consequences of chemotherapy and the effects of hospitalization. In the present study, we employed the two-factor repeated-measure cross-sectional design to determine the effects of chemotherapy and hospitalization on rest-activity (RA) rhythm and QoL of breast cancer patients. Initially, we randomly selected 39 inpatients and 42 outpatients, scheduled to receive six cycles of chemotherapy, from the Regional Cancer Center (RCC), Raipur, India. Finally, 30 patients in each group were included in the current study. We monitored circadian RA rhythm and QoL using wrist actigraphy and QLQ-C30 and QLQ-BR23, respectively, during the 1st (C1), 3rd (C3) and 6th (C6) chemotherapy cycles. Results revealed that with the progression of chemotherapy cycles (from C1 to C6), all rhythm parameters, namely mesor, amplitude, acrophase, rhythm quotient (RQ), circadian quotient (CQ), peak activity (PA), dichotomy index and autocorrelation coefficient, significantly decreased in both cancer in- and outpatients. In both groups of patients and during C1–C6, all functional and global QoL measures of QLQ-C30 and QLQ-BR23 significantly decreased and the symptoms significantly increased, except constipation, body image, sexual functioning and future perspectives in outpatients. The hospitalization exacerbated the problems associated with the RA rhythm and the QoL of the patients. In conclusion, the current study highlighted the negative consequences of hospitalization among inpatients, irrespective of the stage of cancer. We, therefore, recommend that cancer patients should be administered with chemotherapy as outpatients. The proposed protocol might have a covert bearing on the expression of better physiological state leading to satisfactory treatment outcomes.     

Light at Night Co‐distributes with Incident Breast but not Lung Cancer in the Female Population of Israel

Recent studies of shift‐working women have reported that excessive exposure to light at night (LAN) may be a risk factor for breast cancer. However, no studies have yet attempted to examine the co‐distribution of LAN and breast cancer incidence on a population level with the goal to assess the coherence of these earlier findings with population trends. Coherence is one of Hill's “criteria” (actually, viewpoints) for an inference of causality. Nighttime satellite images were used to estimate LAN levels in 147 communities in Israel. Multiple regression analysis was performed to investigate the association between LAN and breast cancer incidence rates and, as a test of the specificity of our method, lung cancer incidence rates in women across localities under the prediction of a link with breast cancer but not lung cancer. After adjusting for several variables available on a population level, such as ethnic makeup, birth rate, population density, and local income level, a strong positive association between LAN intensity and breast cancer rate was revealed (p<0.05), and this association strengthened (p<0.01) when only statistically significant factors were filtered out by stepwise regression analysis. Concurrently, no association was found between LAN intensity and lung cancer rate. These results provide coherence of the previously reported case‐control and cohort studies with the co‐distribution of LAN and breast cancer on a population basis. The analysis yielded an estimated 73% higher breast cancer incidence in the highest LAN exposed communities compared to the lowest LAN exposed communities.     

CD147, CD44, and the Epidermal Growth Factor Receptor (EGFR) Signaling Pathway Cooperate to Regulate Breast Epithelial Cell Invasiveness

The immunoglobulin superfamily glycoprotein CD147 (emmprin; basigin) is associated with an invasive phenotype in various types of cancers, including malignant breast cancer. We showed recently that up-regulation of CD147 in non-transformed, non-invasive breast epithelial cells is sufficient to induce an invasive phenotype characterized by membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invadopodia activity (Grass, G. D., Bratoeva, M., and Toole, B. P. (2012) Regulation of invadopodia formation and activity by CD147. J. Cell Sci. 125, 777–788). Here we found that CD147 induces breast epithelial cell invasiveness by promoting epidermal growth factor receptor (EGFR)-Ras-ERK signaling in a manner dependent on hyaluronan-CD44 interaction. Furthermore, CD147 promotes assembly of signaling complexes containing CD147, CD44, and EGFR in lipid raftlike domains. We also found that oncogenic Ras regulates CD147 expression, hyaluronan synthesis, and formation of CD147-CD44-EGFR complexes, thus forming a positive feedback loop that may amplify invasiveness. Last, we showed that malignant breast cancer cells are heterogeneous in their expression of surface-associated CD147 and that high levels of membrane CD147 correlate with cell surface EGFR and CD44 levels, activated EGFR and ERK1, and activated invadopodia. Future studies should evaluate CD147 as a potential therapeutic target and disease stratification marker in breast cancer.     

Matrix Metalloproteinase 8 (Collagenase 2) Induces the Expression of Interleukins 6 and 8 in Breast Cancer Cells

Matrix metalloproteinase 8 (MMP-8) is a tumor-suppressive protease that cleaves numerous substrates, including matrix proteins and chemokines. In particular, MMP-8 proteolytically activates IL-8 and, thereby, regulates neutrophil chemotaxis in vivo. We explored the effects of expression of either a WT or catalytically inactive (E198A) mutant version of MMP-8 in human breast cancer cell lines. Analysis of serum-free conditioned media from three breast cancer cell lines (MCF-7, SK-BR-3, and MDA-MB-231) expressing WT MMP-8 revealed elevated levels of IL-6 and IL-8. This increase was mirrored at the mRNA level and was dependent on MMP-8 catalytic activity. However, sustained expression of WT MMP-8 by breast cancer cells was non-permissive for long-term growth, as shown by reduced colony formation compared with cells expressing either control vector or E198A mutant MMP-8. In long-term culture of transfected MDA-MB-231 cells, expression of WT but not E198A mutant MMP-8 was lost, with IL-6 and IL-8 levels returning to base line. Rare clonal isolates of MDA-MB-231 cells expressing WT MMP-8 were generated, and these showed constitutively high levels of IL-6 and IL-8, although production of the interleukins was no longer dependent upon MMP-8 activity. These studies support a causal connection between MMP-8 activity and the IL-6/IL-8 network, with an acute response to MMP-8 involving induction of the proinflammatory mediators, which may in part serve to compensate for the deleterious effects of MMP-8 on breast cancer cell growth. This axis may be relevant to the recognized ability of MMP-8 to orchestrate the innate immune system in inflammation in vivo.     

O-GlcNAcylation of Cofilin Promotes Breast Cancer Cell Invasion

O-GlcNAcylation is a post-translational modification that regulates a broad range of nuclear and cytoplasmic proteins and is emerging as a key regulator of various biological processes. Previous studies have shown that increased levels of global O-GlcNAcylation and O-GlcNAc transferase (OGT) are linked to the incidence of metastasis in breast cancer patients, but the molecular basis behind this is not fully known. In this study, we have determined that the actin-binding protein cofilin is O-GlcNAcylated by OGT and mainly, if not completely, mediates OGT modulation of cell mobility. O-GlcNAcylation at Ser-108 of cofilin is required for its proper localization in invadopodia at the leading edge of breast cancer cells during three-dimensional cell invasion. Loss of O-GlcNAcylation of cofilin leads to destabilization of invadopodia and impairs cell invasion, although the actin-severing activity or lamellipodial localization is not affected. Our study provides insights into the mechanism of post-translational modification in fine-tuning the regulation of cofilin activity and suggests its important implications in cancer metastasis.     

Integrin-binding Protein Nischarin Interacts with Tumor Suppressor Liver Kinase B1 (LKB1) to Regulate Cell Migration of Breast Epithelial Cells

Biallelic inactivation of LKB1, a serine/threonine kinase, has been detected in 30% of lung adenocarcinomas, and inhibition of breast tumor growth has been demonstrated. We have identified the tumor suppressor, Nischarin, as a novel binding partner of LKB1. Our mapping analysis shows that the N terminus of Nischarin interacts with amino acids 44–436 of LKB1. Time lapse microscopy and Transwell migration data show that the absence of both Nischarin and LKB1 from an invasive breast cancer cell line (MDA-MB-231) enhances migration as measured by increased distance and speed of migrating cells. Our data suggest that this is a result of elevated PAK1 and LIMK1 phosphorylation. Moreover, the absence of Nischarin and LKB1 increased tumor growth in vivo. Consistent with this, the percentage of S phase cells was increased, as demonstrated by flow cytometry and enhanced cyclin D1. The absence of Nischarin and LKB1 also led to a dramatic increase in the formation of lung metastases. Our studies, for the first time, demonstrate functional interaction between LKB1 and Nischarin to inhibit cell migration and breast tumor progression. Mechanistically, we show that these two proteins together regulate PAK-LIMK-Cofilin and cyclin D1/CDK4 pathways.